1. Field of the Invention
This invention relates to a method for screening for a substance which alters the interaction between GPR120, a G protein-coupled receptor protein, and phospholipase or salts thereof (in particular, a substance which alters GPR120 mediated cell stimulating activity), and a screening kit to be used for such screening.
2. Background Art
Many of physiologically active substances such as hormones and neurotransmitters regulate biological functions through their specific receptor proteins expressed on cell surface membranes. Many of these receptor proteins share a 7-transmembrane structure which couples with trimeric G protein (guanine nucleotide-binding protein) which is present intracellularly and accordingly, are called G-protein coupled receptors (GPCR).
GPCR is expressed on the cell surface of a variety of functional cells, organs and organ parts and activates or suppresses cellular functions by transmitting a signal intracellularly via binding to its regulatory molecule. Accordingly, GPCR plays an important role in a variety of organs and organ parts. It is important to clarify interactions between GPCR and these physiologically active substances for better understanding of biological functions, and for the development of drugs which are closely related thereto. The development of these therapeutic drugs requires efficient screening for GPCR agonists and antagonists, a functional analysis of a receptor protein expressed in a living body, and expression systems of the gene in appropriate cells.
In recent years, the presence of a number of novel genes have been revealed by a random analysis of cDNA sequences shown in EST database and the like, or a comprehensive analysis of genome DNA. GPCRs share a 7-transmembrane domain and also a number of other common sequences. Because of this, novel members of GPCR have been found among a number of those newly discovered genes. Ligands for these novel GPCRs thus discovered are usually unidentified. Identification of ligands and functional analysis for orphan GPCRs whose ligands are not yet identified are believed to be significantly important because these may provide an opportunity for the development of new therapeutic drugs.
In most cases, it is difficult to predict a ligand for each of orphan GPCRs. Ligands for GPCRs include a wide variety of substances such as biological amines, amino acids, nucleic acids and its metabolites, peptides, proteins (for example, hormones, and chemokines), and lipids. Purification of a ligand from extracts requires an extraction method specific to each type of ligand substances. Also, in general, a type of signal transduction system activated by orphan GPCR after responding to a ligand is not easily predictable and studies are required in miscellaneous expression systems. Since prediction of a tissue in which a ligand is present is not easy, a number of different tissue extracts are required. Thus, the ligand identification for orphan GPCRs faces a great deal of difficulty. Discovery of a novel ligand for GPCR and its direct application, or screening for a new drug using the novel ligand is expected to provide an opportunity to develop new drugs of which action mechanism is novel and entirely different from that of currently available drugs.
GPR120 has been known to be one of GPCRs (WO00/00611 and, WO00/50596). Ligands for GPCR120 are not completely elucidated, however, fatty acids has been reported as one of ligands (WO2004/065960 and Japanese Patent Application Laid-Open Publication No. 2005-15358).
GPR120 has been known to be involved in the stimulation of cholecystokinin (CCK) secretion from the STC-1 cell line, an intestinal secretory cell line, and therapeutic applications of GPR120 agonists and antagonists are expected to be useful for eating disorders represented by anorexia and, hyperphagia and intestinal disorders associated with those (Japanese Patent Application Laid-Open Publication No. 2005-15358). In addition, it has been reported that GPR120 has stimulatory effects on the secretion of glucagon like peptide-1 (GLP-1) from the STC-1 cell line, an intestinal secretory cell line, and substances interacting with GPR120 are expected to be useful for its therapeutic application for diabetes (Akira Hirasawa et al., Nature Medicine, 11, 90-94, 2004). Moreover, GPR120 is expressed in the pituitary gland and its potential involvement in stress regulation has been also suggested (WO2004/065960).
As described above, fatty acids have been reported as ligands for GPR120. However, it is often difficult to make a ligand solution with fatty acids because fatty acids are barely soluble in water solvent system. Also, fatty acids are easily absorbed to plastics or glass which is used for screening, and unsaturated fatty acids are easily oxidized. Moreover, fatty acids are known to bind to albumin easily. Under physiological conditions, most of fatty acids are bound to blood albumin and only a small part of fatty acids (1%) exist as free fatty acid. For that reason, inhibitory effects of bovine serum albumin (BSA) on GPR120 activity stimulated by fatty acid have been observed in the screening of a GPR120 ligand using fatty acid, and the necessity of screening in the absence of serum or albumin is reported (for example, Akira Hirasawa et al. Nature Medicine, 11, 90-94, 2004). On the other hand, generally, the screening for a drug using cells and proteins is often carried out in the presence of serum or albumin (BSA and the like) since conditions closer to physiological conditions are required. If the screening is carried out in the absence of serum, a long time culture is usually difficult because of cellular damages in the condition where serum is absent. Therefore, it is necessary that cells are cultured in serum containing media in advance and at the time of the screening the media is replaced by the one that contains no serum, which makes the process more complicated.
Accordingly, it has been expected for a new screening system not using fatty acid directly to screen for GPR120 agonists or antagonists.
Phospholipase is a family of enzymes which hydrolyze an ester linkage of glycerophospholipids, and is classified into phospholipase A1, A2, B, C and D depending on the position of the ester linkage to be hydrolyzed. Phospholipase A2 (PLA2) is further classified into secretory (sPLA2), cytoplasmic (cPLA2) and calcium independent (iPLA2) forms. Among them, there are 10 enzymes known for sPLA2.